The invention relates to a method and a device for determining optical properties by measuring intensities at a thin layer for the detection of chemical, biochemical, medical and/or physical reactions, binding and/or accretion processes as well as other interactions, as preferred uses, in particular in the field of homeland security.
It is known that one can determine physical, chemical, biochemical or biological processes such as reactions, binding and accretion processes and other forms of interaction, at a thin layer of at least partially optically transparent material, by changing the optical layer thickness. For this, light of at least one specific, selected wavelength is radiated onto the sample that is to be examined, which is bound to the thin layer. Interference phenomena are used to determine changes in the optical layer thickness, caused for example by the way a substance that is to be examined reacts with the thin layer that has been subject to an appropriate pre-treatment.
The measurements can be made with the aid of appropriate markers, such as fluorescent markers. More recently, however, they can now also be carried out without markers, and time- and space-resolved.
For the irradiated light, either a single wavelength or several, spectrally separated and thus individual different wavelengths, are radiated onto the thin layers to be examined, and are measured.
Changes in the optical layer thickness are calculated by the spectral location of the interference extrema and their separations from each other. The aim is to observe a shift in the interference pattern. The optical layer thickness can also be determined from the change of intensity at one or more wavelengths. To do this, in prior art one chooses the optimal wavelengths likely to cause the maximum change in light intensity.
WO 2008/067528 A2 D1 describes a so-called “imaging system” at the molecular level, based on the principle of interferometry. Here the analytes in a sample are determined using a measurement assembly which features a light source and a detector for the image capture in the form of a pixel array detector, PAD, with a large number of image elements, so that the irradiated light can be captured and displayed with a good spatial resolution. A biolayer reacts with the analytes that are to be identified, when the sample that is to be examined is brought in contact with it. This biolayer is anchored on a substrate which can convert a phase modulation into an intensity modulation, so that the intensity modulation can then be recorded and displayed directly via the pixel matrix. In addition, a reference surface is provided. First the biolayer is irradiated and the light reflected from the biolayer is forwarded to the pixel matrix, where an image of the sample is produced. Using a so-called image switching unit, which can be a mirror, firstly light is radiated onto the biolayer, and secondly the irradiated light is guided to the reference surface. To do this the mirror is moved accordingly. The light reflected from the reference surface is also forwarded and imaged as the reference image. Using a computer evaluation unit the image of the sample and the reference image are then superimposed. Instead of the mirror the alternating irradiation of the biolayer and the reference layer can also be done using a fast-spinning disk or a polarizing beam splitter.
EP 0 598 341 A1 discloses one or more sensors for the measurement of gaseous and liquid components. The respective optical sensor has a thin film which reacts with the particles to be measured. The measurement is performed via a reflection that is enhanced by interference. The basis for the measurement is the change in layer thickness at the thin film and/or the change in refractive index. The change in intensity of the reflected light is used as the parameter for measurement. If several such sensors are used, they are intended to record various different chemical compounds.
The known methods are very sensitive to intensity variations in the light radiated onto the thin layer. A disadvantage with all previously used methods is that the system technology on which they are based is characterized by a considerable dependence on the intensity. The measuring results in the known methods are directly dependent on the intensity measurements at the region(s) of the thin layer, where changes in the layer thickness were at least partially caused by the interaction with a sample. Because only very small changes in intensity are to be measured here, the measurement of the intensity can be distorted by changes in brightness of the light source. Therefore intensity fluctuations in the region of the incident light have a direct effect on the quality of the measurement results.
The fact that no uniform distribution of intensity could be achieved also had an adverse effect on the reference measurements for the brightness of the light source, at least for such measurements that are to be carried out using so-called multiwell plates. Thus for example for a standard multiwell plate with 96 floors a large common light source in combination with large lenses was used to properly irradiate the multiwell plate, in particular the 96 floors of the multiwell plate. Here it was found that the quality of the light was only sufficient in the central part of the light field emitted by the light source. Therefore the sensitivity and the reliability of these methods of measurement were still inadequate, making it difficult to use the methods in practice.
Basically, the measuring setup for performing such interference measurements consists of a light source, which can be either a xenon high-pressure lamp or an LED (light-emitting diode or a superluminescent diode), a planar carrier, one surface of which is specially activated and pre-treated, and at which the changes in the optical layer thickness are measured, as well as a detector, and an evaluating device
In addition to other disclosures, a technique in prior art is known from WO-A-2006/131225 which describes the details of preparing the planar carrier for performing the interference measurements.
Another technique known in prior art is to be mentioned, in connection with the detection of physical, chemical and/or biochemical reactions and interactions at and/or in samples, where the samples are arranged in a planar shape on a substrate plate that has a carrier layer on a carrier plate. The samples are either irradiated with light of various different wavelengths from a variable frequency light source, or a polychromatic light source is used which is fitted downstream with a scanning monochromator. The irradiation of the light is thus always done in sequence, one wavelength at a time.
The portion of the reflected beam coming off at least one boundary layer surface of every single sample, or the portions of the beam or interference that are reflected off and interfering together at the boundary layer surfaces arrayed one behind the other in the direction of the light, are displayed by optical elements in a space-resolving, planar detector array or a video camera. In particular, WO-A-97/40366 discloses an arrangement that includes a plurality of discrete, photoelectric receivers in the form of CCD elements, which are arranged in a matrix-like pattern and thus provide a spatially resolved planar detector arrangement.
This prior art always involves a selective-wavelength detection of the reflected radiation intensities or intensities of the imaged interference effects affected by the samples, i.e. the detection of a spectrum of wavelengths allocated to each sample, and the resulting derivation of parameters that characterize the interactions and reactions to be investigated, is carried out separately and successively for each wavelength. This requires a great deal of work for the evaluation and a correspondingly large amount of time to derive the desired parameters.
All the measurement setups where the detection of the associated wavelength spectrum of a sample, and the resulting derivation of parameters characterizing the interactions and reactions to be investigated, is done separately and successively for each wavelength, have in common that they entail significant computing work to calculate the changes in layer thickness and the underlying concentration levels. The computational complexity is associated with a significant computing time, which makes an evaluation in real time for many samples that are to be analyzed simultaneously very demanding or indeed no longer technically feasible.
In order to achieve a much faster evaluation combined with a more precise determination of the optical properties in the measurement procedure, which thus allows automated measurements and is suitable for routine measurements, PCT/EP2010/002752 proposes a modified method and a corresponding measuring device. In this method, the determination of the wavelength spectrum associated with each sample and the resulting derivation of parameters characterizing the interactions and reactions to be investigated, is not done separately and successively for each wavelength. Instead, light of a narrow-band spectrum is radiated onto the sample and also evaluated as such, as a whole. The detection of the reflected radiation intensities affected by the samples, or the intensities of the imaged interference patterns, is therefore done using a band of wavelengths. This also means that no direct detection of a wavelength spectrum associated with each sample as a function of only one wavelength is made. Instead all the data are available in combination with at least one lookup table, so that it is possible to call up the information about the optical behavior at one wavelength, and the evaluation can actually be made on the basis of the irradiated narrowband spectrum. In this way the method disclosed in the said PCT application can provide a quick and accurate determination of optical properties at thin layers, which allows an automated measurement and is therefore also suitable for routine use.
However, according to PCT/EP2010/002752 this is only achieved by using a special measuring setup, which depends on the type of light radiated on the carrier bearing the thin layer. So according to PCT/EP2010/002752 the light must be radiated directly onto the carrier. With this direct radiation one cannot use a beam splitter to divert some of the radiated light to be used as a reference.
Starting from this prior art, the present invention therefore had the basic aim of further developing the method and the device presented in PCT/EP2010/002752 so as to allow a quick, automated measurement and thus a routine use, and to ensure that its design for the irradiation of light does not depend on the light being radiated directly onto the carrier plate with the restrictive measures that entails.